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2022-RA-1499-ESGO Differential response of in-vitro mismatch repair-deficient hypermethylated endometrioid endometrial cancer models to DNA-hypomethylating agents
  1. Louis El Khoury1,
  2. Wan Hsin Lin2,
  3. James Smadbeck1,
  4. Dorsay Sadeghian1,
  5. John Cheville1,
  6. Faye Harris1,
  7. Lindsey Kinsella2,
  8. Marina Walther Antonio1,
  9. Giuseppe Cucinella1,
  10. Gabriella Schivardi1,
  11. Alexa McCune1,
  12. Giannoula Karagouga1,
  13. Aaron Mansfield1,
  14. Andrea Mariani1,
  15. George Vasmatzis1,
  16. Panos Anastasiadis2,
  17. John Weroha1 and
  18. Alyssa Larish1
  1. 1Mayo Clinic, Rochester, MN
  2. 2Mayo Clinic, Jacksonville, FL


Introduction/Background We sought to compare in-vitro mismatch-repair deficient endometrial cancer (EC) methylation and responses to DNA-hypomethylating agents using spheroid-based microcancer 3D tumor cell viability assay.

Methodology Study tumor was prospectively collected from a patient with stage 1B, grade 2 endometrioid EC. Characterization entailed whole exome, RNA, and MatePair analysis. Somatic mutations, structural variants and transcriptomic profiling were used to identify potential driver pathways for inhibition. Epigenomic profiling was completed with Assay for Transposase-Accessible Chromatin and DNA-methylation with Reduced Representation Bisulfate Sequencing. A comparative hyper-duplicated, p53-mutated EC underwent identical testing. 3D microcancers of these tumors were subjected to DNA-methyltransferase (DNMT) inhibition. Cell viability was determined by CellTiter-Glow Luminescent Assay. Data transformation and dose-response curves were generated by GraphPad Prism using four-parameter logistic regression. Inhibitory effect (IE) was defined as percent reduction ATP from baseline at maximum plasma concentration (Cmax).

Results Genomic sequencing revealed evidence of microsatellite instability with POLE variant of unknown significance. Global and promoter hypermethylation was observed in sample with fewer copy number variation. When contrasted with comparison tumor, we observed significant (p < 0.01), albeit modest, global (Δβ = 0.51) and promoter (Δβ = 0.52) hypermethylation. Methylation of both MLH1 and PMS2 was observed. While both gene bodies were hypermethylated (Δβ = 0.50 and Δβ = 0.15 respectively), only MLH1 was statistically different. Despite the lack of methylation of promoters for both genes, we noticed a gene expression fold reduction of 2.58 (MLH1) and 1.81 (PMS2). Inhibition of viability in both study and comparison was minimal by decitabine, shown by IE of 0 and 17.939, respectively. Conversely, IE of study tumor by azacitidine was more pronounced at 72.662, compared with 40.951 (figure 1).

Abstract 2022-RA-1499-ESGO Figure 1

Normalized drug responses of study tumor (pink) and comparison tumor (blue) to azacitidine and decitabine. Dose-response curves of treatment were titrated for each agent across maximum inhibitory plasma concentration (black dotted line). Pink and blue dotted lines represent results of previous testing

Conclusion In MMR-D EC with MLH-1 hypermethylation, in-vitro tumor response to DNMT inhibition is superior for DNA/RNA incorporating azacitidine when compared to DNA-only incorporating decitabine.

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